The project continues our structural studies of the two key regulatory enzymes of carbohydrate metabolism, phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FbPase). The research, which involves a major collaborative effort of three laboratories, includes the following: (1) The primary sequence of rabbit muscle PFK, which is almost complete, will be finished and predictions concerning three dimensional organization will be tested by cross-linking experiments. (2) Sequences around functional residues of muscle PFK will be determined by covalent modification techniques. (3) Predictions concerning the evolution of allosteric sites of PFK will be evaluated by functional site identification and partial sequence analysis of E. coli and yeast (Saccharomyces cerevisiae) PFK. (4) Functional site identification and sequence analysis of E. coli FbPase, and an analysis of the phosphorylation site(s) of Saccharomyces cerevisiae FbPase will be compared to data obtained with mammalian FbPases. (5) The sequence of skeletal muscle FbPase, an enzyme with increased sensitivity to AMP inhibition, will be determined and compared to the kidney and liver enzyme. These studies provide structural correlates to our knowledge of allosteric interactions and is of interest to our overall understanding of enzyme regulatory mechanisms.